The Fibroblast Growth Factor (FGF) gene superfamily is a family of conserved, secreted proteins that have been shown to play a critical role in many biological processes (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). FGF-signalling is achieved by binding of the ligand, FGF, to the extra-cellular domain of high affinity membrane bound FGFR, which belongs to the tyrosine kinase family of receptors (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). Today around 20 different FGF genes and 4 different FGFR genes have been identified, and multiple ligands can interact with one and the same receptor (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). The level of complexity of signalling via these receptors is further compounded by the fact that alternative splice variants exist for these receptors. Loop three of the extracellular domain (=ligand binding domain) can splice to give rise to b, or c, isoform. This isoform variation ultimately determines ligand specificity and proper ligand-receptor interaction ultimately leads to activation of the intracellular tyrosine kinase domain (Kato and Sekine, 1999; Szebenyi and Fallon, 1999).
FGF-signalling has been implicated in a variety of distinct biological processes including patterning, differentiation, morphogenesis, proliferation, survival, angiogenesis, tumorogenesis, etc. (Kato and Sekine, 1999; Szebenyi and Fallon, 1999). In mouse, an early embryonic lethality or functional redundancy have, however, largely hampered direct genetic approaches aiming at elucidating the role of FGF-signalling during development and in the adult. Thus these approaches has for the most part failed to provide critical information regarding the role of FGF-signalling during later stages of vertebrate organogenesis, including the pancreas. An alternative approach have been to impair FGF-signalling via organ specific expression of dominant negative forms of FGFR that will competitively block FGF signalling via the endogenous, corresponding FGFR variant. This approach has been successfully used to antagonise FGF-signalling in a number of different systems.
Viral infection of a dominant negative FGFR1 construct in chick limb muscle mass blocked the differentiation of myoblast to myotubes providing evidence that this process depends on FGF-signalling (Itoh et al., 1996). Studies focused on maintenance of cell types within the retina revealed that expression of dnFGFR2 under the control of the bovine rhodopsin promoter increased photoreceptor degeneration (Campochiaro et al. 1996). The specificity of dominant negative constructs with respect to ligand binding was demonstrated in analyses where dnFGFR1c and dnFGFR2b constructs where expressed in transgenic mice using the mammary tumour virus promoter (Jackson et al.,1997). Expression of dnFGFR1c under these conditions did not result in any discernible phenotype whereas an impairment of lobuloalveolar development in the mammary gland was observed when using the dnFGFR2b variant (Jackson et al.,1997). Moreover, FGF8 mediated induction of dopaminergic (DA) neurons was successfully inhibited when growing six somite rat ventral mid/hindbrain explants in presence of soluble dnFGFR3c, i.e. the high-affinity blocking receptor for FGF8 (Ye et al., 1998). In contrast, when the same experiment was performed using soluble, dnGFGR1c, a low-affinity nonblocking receptor for FGF8, DA neurons readily appeared (Ye et al., 1998). Together these analyses demonstrate the effectiveness by which FGF signalling, in an apparent ligand-specific manner, can be perturbed using a dominant-negative FGFR approach.
Three different FGF-signalling mutant mice, involving transgenic approaches to over-express either a ligand or a dn form of a receptor, resulting in a pancreatic phenotypes have been reported. Transgenic over-expression of FGF7/KGF in the mouse liver induced pancreatic ductual hyperplasia (Nguyen et al. 1996) and similarly, transgenic mice with forced expression of FGF-7/KGF in pancreatic β-cells under the control of the insulin promoter show enlarged islets containing proliferating duct cells (Krakowski et al., 1999). General transgenic over-expression of dnFGFR2b under the control of the metallothionein promoter resulted in pancreatic hypoplasia (Celli et al. 1998). Together these studies indicate that signalling through FGFR2b may operate during pancreatic development. In vitro experiments involving culturing of pancreatic rudiments support such a scenario and suggest that FGFs positively stimulate pancreatic epithelial cell proliferation and exocrine cell differentiation (Le Bras et al. 1998, Miralles et al. 1999).
Selective inactivation of the IIIb form of FGFR2 leads to developmental abnormalities in limbs, lung, anterior pituitary, salivary glands, inner ear, teeth and skin but apparently not in the pancreas (De Moerlooze et al., 2000). Thus, the roles of FGFR2b during pancreas development remain to be determined.
Failure of the β-cell to compensate for an increased demand for insulin is a key feature in the manifestation of type 2 diabetes. Type 2 diabetes is the most common form of diabetes, affecting 2–3% of the world-wide population, and is the combined result of resistance to insulin action coupled with a defect in β-cell compensation (Kahn, 1998; Kahn and Rossetti, 1998; Taylor, 1999). The molecular defects underlying the development of the disease are not fully understood and there are also uncertainties as to what is the primary defect initiating the disease; the insulin resistance or the β-cell failure. A typical trait associated with the disease is the increased proinsulin to insulin (P/I) ratio observed in many type 2 diabetic patients (Porte and Kahn, 1989). The relationship between the increased P/I ratio and the etiology of the disease has however remained diffuse; i.e. is it a consequence rather than a directly contributing factor to the disease? Several independent studies points towards an increased P/I ratio being an early sign of primary β-cell dysfunction, independent of insulin resistance, which is directly associated with the conversion from a prediabetic to an overt diabetic state over a short time period (Mykkänen et al., 1995; Kahn et al., 1995, Nijpels et al., 1996; Rachman et al., 1997; Mykkänen et al., 1997; Haffner et al 1997; Larsson and Ahrén, 1999). Moreover, it has been suggested that normal β-cells respond to an increased insulin resistance by enhanced processing of insulin and that the increased P/I ratio in individuals with an impaired glucose tolerance, and/or type 2 diabetes, is the consequence of defects in proinsulin processing (Mykkänen et al., 1997, Larsson and Ahrén, 1999).
Processing of proinsulin to insulin in β-cells is catalysed by the sequential actions of prohormone convertases PC1/3 and PC2, which both act in concert with carboxpeptidase E (CPE) (FIG. 7) Analyses of PC2 null mutant mice demonstrated a crucial role for PC2 in the processing of proglucagon and prosomatostatin in α- and δ- cells, respectively (Furuta et al., 1998). Proinsulin processing in β-cells was less affected in the PC2 null mutant mice providing evidence that PC3 is quantitatively more important than PC2 with respect to processing of proinsulin to active insulin (Furuta et al., 1998). At present there is a lack of genetically defined animal models that mimic these aspects of human type 2 diabetes. The importance of this disease in terms of human suffering and health care costs makes the provision of such a model an important goal.